The development of an effective prophylactic vaccine for HIV-1 will likely need an immunogen that can induce neutralizing antibody, and CD4 and CD8 T cell activity. Viral vector systems that infect cells and allow intracellular expression of HIV-1 gene products have the ability to activate T cells through MHC class I and II presentation. Herpes simplex virus type-1 (HSV-1) amplicons possess many of the desirable features of such a viral vector system. They are non-replicating, induce robust CD8+ T cell responses in mice, are easily manufactured, and infect a variety of antigen presenting cells, including dendritic cells. The HSV-1 amplicon can incorporate large segments of DNA, express more than one gene product, are not contaminated by helper virus, and are under development and evaluation as gene therapy tools. In initial experiments, HSV-1 amplicons expressing HIV-1 MN gp120 were shown capable of inducing interferon gamma-producing T cells at a number equivalent to that induced by live herpesvirus vectors, and far exceeding that of a modified vaccinia Ankara vector. In addition, the amplicons induced large anti-Env antibody responses. Building upon these observations, three specific aims are proposed. The first will be to construct amplicons which express codonoptimized clade C env, gag, and tat genes, and evaluate the protein expression from such vectors in vitro. In the second aim, the optimum route of parenteral and mucosal delivery, the dose and duration of immunity, and the effect of prior immunity to HSV will be evaluated. Lastly, the immune responses induced by the clade C amplicon will be evaluated in BALB/c and mapped in HLA-A2/human beta2 microglobulin transgenic mice. Overall, it is anticipated that these experiments will generate sufficient data to warrant moving the HSV-1 amplicon vaccine concept into non-human primate and human vaccine trials.